Led driven plasmonic heating apparatus for nucleic acids amplification

ABSTRACT

Systems and methods for plasmonic heating by combined use of thin plasmonic film-based 2D and 3D structures and a light-emitting diode (LED) for nucleic acids amplification through fast thermal cycling of polymerase chain reaction (PCR) are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/649,328 filed on Jul. 13, 2017, incorporated herein by reference inits entirety, which is a 35 U.S.C. § 111(a) continuation of PCTinternational application number PCT/US2016/013732 filed on Jan. 15,2016, incorporated herein by reference in its entirety, which claimspriority to, and the benefit of, U.S. provisional patent applicationSer. No. 62/104,574 filed on Jan. 16, 2015, incorporated herein byreference in its entirety. Priority is claimed to each of the foregoingapplications.

The above-referenced PCT international application was published as PCTInternational Publication No. WO 2016/115542 on Jul. 21, 2016, whichpublication is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

A portion of the material in this patent document is subject tocopyright protection under the copyright laws of the United States andof other countries. The owner of the copyright rights has no objectionto the facsimile reproduction by anyone of the patent document or thepatent disclosure, as it appears in the United States Patent andTrademark Office publicly available file or records, but otherwisereserves all copyright rights whatsoever. The copyright owner does nothereby waive any of its rights to have this patent document maintainedin secrecy, including without limitation its rights pursuant to 37C.F.R. § 1.14.

BACKGROUND 1. Technical Field

This description pertains generally to systems and methods forpolymerase chain reaction (PCR) diagnostics, and more particularly toplasmonic heating systems and methods for fast/ultrafast PCR.

2. Background Discussion

After its initial introduction in 1983, polymerase chain reaction (PCR)has become an essential technique in the field of clinical laboratories,agricultural science, environmental science, and forensic science. PCRuses thermal cycling, or repeated temperature changes between 2 or 3discrete temperatures to amplify specific nucleic acid target sequences.To achieve such thermal cycling, conventional bench-top thermal cyclersgenerally use the metal heating block powered by Peltier elements. Whilecommercial PCR systems are improving heating and cooling rates to reduceamplification time, they are still relatively time-consuming (typicallyrequiring an hour or more per amplification). This can be attributed tothe larger thermal capacitance of a system that requires uniform heating96- or 384-well plastic PCR plates and reaction volumes of several tensof microliters per well.

Since fast/ultrafast PCR is highly desirable for applications such astimely diagnosis of infectious diseases, cardiac diseases, cancer,neurological disorder diseases, and rapid biowarfare and pathogenidentification at the point-of-care (POC) level, many academic andindustrial groups have been improving PCR systems. However, existingsystems are not suitable for POC testing due to high power consumption,heavy weight require a complicated fabrication process, prone to humanerror, require expensive lasers and detection systems, or havereliability issues.

Accordingly, an object of the present description is a fast/ultrafastPCR system that is portable, robust, simple, easy to use andcharacterized by low power consumption through miniaturization andintegration.

BRIEF SUMMARY

The present description is directed to systems and methods for nucleicacid amplification and quantification via polymerase chain reaction(PCR) for clinical laboratories, precision medicine, personalizedmedicine, agricultural science, forensic science, and environmentalscience. Ultrafast multiplex PCR, characterized by low powerconsumption, compact size and simple operation, is ideal for timelydiagnosis at the point-of-care (POC).

One aspect is an ultrafast photonic PCR method using plasmonicphotothermal light-to-heat conversion via photon-electron-phononcoupling. An efficient photonic heat converter is disclosed, using athin gold (Au) film for its plasmon-assisted high optical absorption(˜65% at 450 nm, peak wavelength of heat source LEDs). Theplasmon-excited Au film is capable of rapidly heating the surroundingsolution to over 150° C. within 3 min. Using this method, ultrafastthermal cycling (e.g. 30 cycles; heating and cooling rate of 12.79±0.93°C. sec⁻¹ and 6.6±0.29° C. sec⁻¹, respectively) from 55° C. (temperatureof annealing) to 95° C. (temperature of denaturation) is accomplishedwithin 5 minutes. Using photonic PCR thermal cycles, we demonstrate heresuccessful nucleic acid (λ-DNA) amplification. Our simple, robust andlow cost-approach to ultrafast PCR using an efficient photonic-basedheating procedure could be generally integrated into a variety ofdevices or procedures, including on-chip thermal lysis and heating forisothermal amplifications.

Thin Au films with nanometer sized grain prepared by electron beamevaporation are characterized to enhance light absorption throughsurface plasmon resonance, leading to fast plasmonic heating of thin Aufilm. Low cost (<$5) LED can effectively increase temperature of liquidup to 140° C. by focusing light with low cost lens (<$1). The maximumramp rate is up to 7° C./sec and temperature stability at 50° C. and 90°C. are ±0.5° C. and ±0.7° C., respectively. Nucleic acids amplificationsthrough fast thermal cycling (from 50° C. to 90° C., 40 cycles for 19min) for PCR are successfully demonstrated using a LED driven plasmonicheating of thin Au films. With this invention, we can obtain simple,portable PCR thermal cycler with extremely low cost and powerconsumption for point-of-care diagnostics.

The technology could be easily used for fast thermal cycler for nucleicacids detection using PCR and/or isothermal amplification including LAMPand RPA. Fast thermal cycler for PCR nucleic acids detection would bethe best ways to utilize the disclosed invention.

In general, the invention will be used for fast thermal cycling fornucleic acids amplifications using polymerase chain reaction (PCR),loop-mediated isothermal amplification (LAMP), and recombinasepolymerase amplification (RPA) in point-of-care testing for human,animal healthcare or environmental.

The most distinctive advantage is to obtain low cost (LEDs <$5, focuslens <$1) and low power consumption (LEDs <3 W) thermal cycler withsimple plasmonic heater fabrication (complicated patterning process orexpensive gold nanoparticles are not required). Furthermore, itssimplicity and easy system level integration for point-of-care nucleicacids testing are other advantages.

Further aspects of the technology will be brought out in the followingportions of the specification, wherein the detailed description is forthe purpose of fully disclosing preferred embodiments of the technologywithout placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The technology described herein will be more fully understood byreference to the following drawings which are for illustrative purposesonly:

FIG. 1 shows a schematic diagram illustrating the principle of ultrafastphotonic PCR in accordance with the technology of the presentdescription.

FIG. 2A and FIG. 2B show a schematic diagram of a system for ultrafastphotonic PCR using thin gold (Au) film as a light-to-heat converter andexcitation light from the light-emitting diodes

FIG. 3 shows a schematic diagram of a LED-driven ultrafast photonic PCRsystem.

FIG. 4A through FIG. 4F show simulation for heat generation of thin Aufilms of the present description with electromagnetic radiation. FIG. 4Aand FIG. 4B show images for electromagnetic field on 10 nm and 120 nmthick Au films on PMMA substrate, respectively. FIG. 4C and FIG. 4D showimages for resistive heat distributions on 10 nm and 120 nm thick Aufilms on PMMA substrate, respectively. FIG. 4E shows a plot ofcalculated absorption spectra of the thin Au films with differentthickness. FIG. 4F shows a plot of light-to-heat conversion efficiencyof the thin Au films averaged over emission wavelength from 3 differentcolored LEDs as a function of Au films thickness.

FIG. 5A through FIG. 5F show plots of LED driven photothermal heating ofthin Au film and PCR thermal cycling.

FIG. 6A is a plot of representative temperature profiles of 30 ultrafastphotonic PCR thermal cycles from 95° C. (denaturation) to 55° C.(annealing and extension).

FIG. 6B shows a plot of heating and cooling rates obtained from theultrafast photonic thermal cycling.

FIG. 6C is an image showing formation of product from the photonic PCRthermal cycler in comparison with bench-top thermal cycler using λ DNAtemplate.

FIG. 7 is a cross-section of representative geometry used for thesimulation of electromagnetic field and resistive heat distribution inthin films.

FIG. 8 is a plot of normalized light emission spectra measured from 3different LEDs with peak wavelengths of 450 nm (Blue I), 480 nm (BlueII) and 530 nm (Green), respectively.

FIG. 9A and FIG. 9B are plots of transmittance and reflectance spectra,respectively, of the thin Au films on PMMA substrate with differentthicknesses.

FIG. 10 is a plot comparison of 31 ultrafast thermal cycles from 62° C.to 94° C. with and without cooling fan.

FIG. 11 is a schematic diagram of an experimental set up for LED drivenplasmonic heating of thin Au films.

FIG. 12 is a plot showing fast thermal cycling using LED drivenplasmonic heating of thin Au films.

FIG. 13A is a plot showing temperature changes of liquid (glycerol, 5μL) with different thin Au films thickness. Injection current of LEDswas fixed at 700 mA.

FIG. 13B shows a plot temperature for varying the film 20 thickness.

FIG. 13C is a plot showing absorbance changes of thin Au films as afunction of wavelength. The inset shows the representative SEM images ofthin Au films containing nanometer sized grain.

FIG. 13D and FIG. 13E show images of the Au film 20 at 56 nm and 96 nm,respectively.

FIG. 14A is a plot of temperature changes of liquid (glycerol, 5 μL)with different injection current of LEDs.

FIG. 14B is a plot illustrating changes of maximum temperature (leftaxis) and ramp rate (right axis) as a function of injection current.

FIG. 15 is a plot of temperature stability of LED driven plasmonicheating of thin Au films at 50° C. and 90° C.

FIG. 16 is a plot of the temperature profiles of thermal cyclingmeasured by IR camera during PCR reaction.

FIG. 17 is a photograph of gel agarose gel demonstrating the formationof product from a plasmonic and bench top thermal cycler.

DETAILED DESCRIPTION

Referring to FIG. 1 through FIG. 3, a novel ultrafast photonic PCRsystem and method are shown, combining the use of thin Au film as alight-to-heat converter and a light source such as light-emitting diodes(LEDs) as a heat source.

FIG. 1 shows a schematic diagram of the plasmonic photothermallight-to-heat conversion and subsequent heating of surrounding mediumthrough the ultrafast photon-electron-phonon couplings in accordancewith the technology of the present description. Light (photon) 10 isdirected toward the Au molecule 12 to generate photon coupling 14 andresultant heat generation of the medium 16 (PCR mixture).

In considering photon interaction with materials, the absorption ofphotons is often treated as heat. When the photons 10 from theexcitation source reaches the surface of thin Au molecule 12,plasmon-assisted strong light absorption can occur. This in turn exciteselectrons near the surface to higher energy states, generating hotelectrons within 100 fs. These hot electrons can reach a temperature ofseveral thousand degrees Kelvin due to their small electronic heatcapacity. They are also capable of rapidly diffusing throughout the thinAu film, creating a uniform distribution of hot electrons. Rapid heatingis followed by cooling to equilibrium by energy exchange between the hotelectrons and the lattice phonons after 5˜10 ps. Thus, overall, when theAu is illuminated, a large temperature difference between the hot metalsurface and the cooler surrounding solution 16 occurs, resulting in theheating 14 of the surrounding solution 16 in a long time scale over 100ps.

FIG. 2A and FIG. 2B (exploded view) show a schematic diagram of a system30 for ultrafast photonic PCR using thin plasmonic (e.g. gold (Au)) film20 as a light-to-heat converter and excitation light 10 from a lightsource 22 (e.g., LEDs). In one embodiment, the thin films 20 arefabricated with nanometer sized grain prepared by electron beamevaporation, and are configured to enhance light absorption throughsurface plasmon resonance, leading to fast plasmonic heating of thin Aufilm.

While the plasmonic thin film 20 is detailed throughout the descriptionas being Au, it is appreciated that such selection of materials is forexemplary purposes only, and any number of plasmonic materials may beselected for plasmonic heating of the sample solution 16. For example,the plasmonic thin film 20 may comprise gold (Au), silver (Ag),palladium (Pd), platinum (Pt), nickel (Ni), titanium (Ti), chromium(Cr), germanium (Ge), tungsten (W), iridium (Ir), etc., or anycombination or alloy thereof. The plasmonic thin film can be multi-layermetallic structure composed of the gold (Au), silver (Ag), palladium(Pd), platinum (Pt), nickel (Ni), titanium (Ti), chromium (Cr),germanium (Ge), tungsten (W), iridium (Ir), etc., or any combination oralloy thereof. Furthermore, the plasmonic thin film can be graphene,graphene oxide, graphite, or carbon nanotubes (CNTs), or plasmonic thinfilm can be graphene, graphene oxide, graphite, or carbon nanotubes(CNTs), or a hybrid or materials composed thereof.

Furthermore, while the light source 22 is detailed throughout thedescription as being one or more LED's, it is appreciated that suchselection is made for exemplary purposes only, and any number ofdifferent light sources may be selected for illumination of theplasmonic thin film 20. For example, the light source 22 may compriseLEDs, diode lasers, a diode laser array, a quantum well(vertical)-cavity laser, or combination or array thereof. Additionally,the emission wavelength of light source may be an ultraviolet (UV),visible, or infrared (IR), etc.

As seen in the exploded detail of FIG. 2B, the PCR mixture 16 issubjected to a three-state thermal cycling, comprising of 2 or 3discrete temperatures for denaturation (phase 1), and annealing andextension (phase 2), with resulting in the nucleic acid amplificationphase (phase 3) through the polymerase chain reaction (PCR). As furtherillustrated in FIG. 2, an array of LED's 22 may be disposed on substrate28 for illuminating arrays of PCR wells 24 that are disposed intransparent or translucent platform 26. For a multiple PCR reaction asdepicted in FIG. 2, each LED 22 may be modulated separately to haveunique annealing temperatures for each primer design.

The thin Au film 20 deposited within wells 24 is used as a light-to-heatconverter, serving as a source of plasmonic (i.e. plasmon-excitable)photothermal heating for the PCR thermal cycling as shown in FIG. 2.

Besides driving multiple PCR reactions with single LEDs, multiple wellplates integrated with LED arrays may be used for multiplexed PCR bymodulating each LED 22 to have unique annealing temperatures for thevarious primer designs. Such multiple well LED array PCR thermal cyclerconfiguration is ideal for multiplexed ultrafast PCR at POC diagnostics,because it could perform multiple tests at once.

FIG. 3 shows a schematic diagram of a LED-driven ultrafast photonic PCRsystem 40. Continuous-wave light from blue LEDs 22 is focused on aplasmonic Au thin-film 20 (deposited on bottom of PCR wells 24) throughthe focus lens 32. Lens 32 may include a configured to produce an evenlydistributed light exposure of the plasmonic thin film 20 to light fromthe light source the peak wavelength of the LEDs was 450 nm. (Blue LEDI). The Au-coated PCR wells 24 were formed in a polymeric (e.g. poly(methyl methacrylate) (PMMA) platform 26.

Platform 26 preferably comprises a transparent or translucentcomposition to allow light to pass through to the thin film 20. Whilethe platform 26 is detailed throughout the description as generallycomprising PMMA, it is appreciated that such selection of materials isfor exemplary purposes only, and any number of polymeric ortranslucent/transparent materials may be selected for use as theplatform. The support platform 26 may also comprise 2D or 3Dmicrostructures or nanostructures that may comprise one or more of apillar array, 1D or 2D grating, photonic crystal, hemi-sphere, or otherpatterned or random structures. In one embodiment, the platformcomprises nanoplasmonic structures or nanoplasmonic feedback lasercavity on the surface of the wells the are configured to be illuminatedat a resonance wavelength of nanoplasmonic structures and duration thatcauses plasmonic photothermal heating of the nanoplasmonic structures

A temperature sensor 34 is coupled or directed at the platform 26 formeasuring the temperature of the sample 16 and/or thin film 20. Suchtemperature sensor 34 may comprise a number of possible sensor types,such as thermocouple or camera (e.g. IR camera) directed at the platform26.

It is also appreciated that PCR system 40 may be integrated orcompatible with a diagnostic device, such as digital camera, photodiode,spectrophotometer or the like imaging device (not shown, but may be inplace of or integrated with of IR camera 52 shown in FIG. 11) for thereal-time detection of nucleic acids and/or the fluorescence signal ofthe sample solution 16. In some configurations the camera may be smartphone camera, wherein the smart phone comprises application software foranalysis of the sample solution 16.

In a preferred embodiment, the sensor and LED's 22 may be coupled to acomputing unit 42 for acquisition of sensor data and control of theLED's 22. Computing unit 42 generally comprises a processor 44, andmemory 46 for storing application software 48 executable on theprocessor 44 for driving the LED 22 (e.g. controlling LED timing,intensity/injection current, etc.), acquiring data from sensor 34 and/orprocessing data from a diagnostic device such as a digital camerareal-time detection of nucleic acids and/or the fluorescence signal ofthe sample solution 16. Computing unit 42 may comprise a separatecomputer or device, or may be integrated into a microcontroller modulewith the remainder of the components. Acquired data and/or a userinterface may be output on a display (not shown) integrated with orcoupled to the computing unit 42.

In one embodiment, strong light absorption of the thin Au film 22 (e.g.65%, 120 nm thick) generates heat due to the plasmonic photothermallight-to-heat conversion by photon-electron-phonon coupling at the thinAu film 20, followed by heating of surrounding solution 16 with amaximum temperature of over 150° C. within 3 min. Ultrafast 30 thermalcycles (heating rate of 12.79±0.93° C. sec⁻¹ and cooling rate of6.6±0.29° C. sec⁻¹) from 55° C. (point of annealing) to 95° C. (point ofdenaturation) are accomplished within 5 min for successful amplificationof λ-DNA.

The PCR systems shown in FIG. 1 through FIG. 3 are ideal for POCdiagnostics, due to ultrafast thermal cycling capability, multiplexedPCR, low power consumption for the PCR thermal cycling (in currentset-up, up to ˜3.5 W), low cost and simple configuration for systemlevel integration. Furthermore, the photonic-based heating procedure ofthe present description may be generally integrated into a variety ofdevices or procedures, including on-chip thermal lysis and heating forisothermal amplifications.

Example 1

a. Fabrication

Several 4 mm-thick poly(methyl methacrylate) (PMMA) sheets 26 were cutwith a VersaLASER VL-200 laser cutting system (Universal Laser System,Inc.) to make reaction wells 24 with a 4 mm diameter. A 1.5 mm-thickbottom PMMA sheet were attached to the top sheet containing reactionwells were bonded together using the thermal bonding. Thermal bondingwas performed at 84° C. with pressure of 1.0 metric ton after UV/Ozonetreatment of PMMA sheet for 10 min. The thin Au films 20 of differentthicknesses were deposited by electron beam evaporation under basepressure of 2×10⁻⁷ Torr. The thin Au film 20 was then passivated withthin ploy(dimethylsiloxane) (PDMS) by dropping 3 μL of PDMS into thewell and curing in the oven for 2 hrs to prevent PCR reaction inhibitionby the thin Au film and thermocouple.

b. Simulation

COMSOL Multiphysics software (Ver. 4.3) was used for performingsimulations. The detailed geometry and materials properties forsimulation are shown in FIG. 7 and Table 1. A thin Au film was placed ona PMMA substrate and water was disposed on the top of Au film. Differentthicknesses (10 nm, 20 nm, 40 nm, 80 nm, and 120 nm) of thin Au filmwere applied to the simulation to calculate the absorption of the Aufilms and subsequent resistive heat generation. The plane wave withx-polarized electric field travels in the positive z direction in thecoordinate shown in FIG. 7. The permittivities of PMMA and water were 3and 1.77, respectively.

A set of electromagnetic simulations was performed to theoreticallycharacterize the plasmonic photothermal light-to-heat conversion of theAu films. The electromagnetic (EM) field and resistive heatdistributions were calculated for 10 nm and 120 nm thick Au films on aPMMA substrate. FIG. 4A and FIG. 4B show images for electromagneticfield on 10 nm and 120 nm thick Au films on PMMA substrate,respectively. FIG. 4C and FIG. 4D show images for resistive heatdistributions on 10 nm and 120 nm thick Au films on PMMA substrate,respectively. FIG. 4E shows a plot of calculated absorption spectra ofthe thin Au films with different thickness. FIG. 4F shows a plot oflight-to-heat conversion efficiency of the thin Au films averaged overemission wavelength from 3 different LEDs as a function of Au filmsthickness.

As expected from skin depth,

$\delta = \sqrt{\frac{2}{\omega\mu\sigma}}$

where ω: angular frequency, μ: permeability, σ: conductivity, thethickness of thin Au film determines the amount of light to heatconversion. Upon a normal incidence of a 450 nm wavelength light source,the 10 nm thick Au film transmits an enormous amount of EM energy (FIG.4A), and the heat conversion energy is saturated along the film depth(FIG. 4C). However, the 120 nm thick Au film absorbs most of theincident light (FIG. 4B) and subsequently generates more heat in the Aufilm by converting light into heat (FIG. 4D).

FIG. 4E shows a plot of calculated absorption spectra of the thin Aufilms with different thickness, Light-to-heat conversion efficiency ofthe thin Au films averaged over emission wavelength from 3 differentLEDs as a function of Au films thickness. The blue LED with 450 nm peakwavelength shows the highest averaged light-to-heat conversionefficiency, illustrating that an increase in thickness of thin Au film,in the range of 10 to 120 nm, corresponds to an increase in opticalabsorption. Significant increase of optical absorption below 540 nmwavelength could be attributed to the plasmonic electron resonance ofgold. As a result, the averaged light-to-heat conversion efficiency overemission wavelength from each LED increases with increased Au filmthickness for the 3 different LEDs (Blue I at 450nm, Blue II at 480nm,and Green at 530 nm). as shown in FIG. 4F (see also FIG. 8). It isnoteworthy that the blue LEDs with a peak emission wavelength of 450 nmshows highest light-to-heat conversion efficiency with thin Au film.

c. Test Setup

A test setup similar to the system 40 shown in FIG. 3 was used forexperimentation. LEDs 22 (Luxeon Rebel royal blue star LEDs with a peakwavelength of 447.5 nm, 890 mW at 700 mA injection current) were usedfor plasmonic photothermal heating of the thin Au film 20 with aKeithley 2400 source meter (not shown). To focus the light from theLEDs, a Carclo 20 mm fiber coupling optic 32 was employed. Thetemperature of the solution was monitored and recorded in real time by atype-K insulated thermocouple (OMEGA Engineering) for thermal cycling.Temperature cycling using an LED, 80 mm cooling fan (not shown), sourcemeter and thermocouple was controlled through LabVIEW as the applicationsoftware. A National Instruments (NI) 16 channel thermocouple module(not shown) with high speed mode, auto zero and cold junctioncompensation (CJC) was used for accurate temperature acquisition fromthe type-K thermocouple.

d. Preparation of the PCR Reagent and DNA Template

A template λ-DNA and Takara Z-Taq DNA polymerase (2.5 U/μL), 10× Z-TaqBuffer (Mg²⁺ plus, 30 mM) and dNTP Mixture (2.5 mM each) were used.Forward primer and reverse primer were purchased from Integrated DNATechnologies. The reactions used to amplify a 98 base pair (bp) λ-DNAtarget with Z-Taq DNA polymerase included 0.5 μL Z-Taq DNA polymerase, 5μL of 10× Z-Taq Buffer, 4 μL of dNTP mixture, 4.5 μL of 10 μM primers(each), 10 μL of bovine serum albumin (BSA) (50 μg), and was brought to50 μL with PCR grade water. The final concentration of template λ-DNAwas 0.1 ng/μL. The 10 μL of PCR mixture was placed within an Au-coatedPMMA PCR wells for photonic PCR, and then covered with 30 μL of mineraloil to prevent evaporation during thermal cycling. After amplification,the mixture of 10 μL of PCR product and 10 μL of E-Gel sample loadingbuffer (Invitrogen) was loaded onto E-Gel 2% agarose gels with SYBR Safe(Invitrogen), and ran in an E-Gel base (Invitrogen) for 30 min. A 1 KbDNA ladder was used to confirm the size of product.

e. LED-Driven Photonic PCR Thermal Cycler

FIG. 5A through FIG. 5F show plots of LED driven photothermal heating ofthe thin Au film and PCR thermal cycling. FIG. 5A shows absorptionspectra of the thin Au films with different thickness. Absorption(%)=100−Transmittance(%)−Reflectance(%). FIG. 5B shows temperature profiles ofliquids as a function of Au film thickness at a 500 mA injectioncurrent. FIG. 5C shows temperature profiles of liquids as a function ofinjection current of LEDs with 120 nm-thick Au film. The blue LED with450 nm peak wavelength was used. FIG. 5D shows a 2D map showing thedistribution of liquid temperature with different thickness of the thinAu film and injection current of LEDs after heating for 3 min. FIG. 5Eillustrates LED driven photonic PCR thermal cycling of 3 differenttemperatures: 94° C. (denaturation), 60° C. (annealing) and 72° C.(extension). FIG. 5F shows temperature control and low temperaturevariations for PCR thermal cycling. Average values with standarddeviation at 94° C., 60° C. and 72° C. were 94.09±0.17° C., 59.94±0.13°C. and 72.02±0.12° C., respectively.

Referring to FIG. 5A, the optical absorption spectra of thin Au filmswith different thicknesses deposited on PMMA substrate are shown. Thesimulation results can help determine when the strongest lightabsorption occurs, as this is critical to maximizing photothermalheating. As the thickness of thin Au film increases, the opticalabsorption also increases, showing 65% absorption at the peak wavelength(450 nm) of excitation LEDs in the 120 nm-thick Au film.

With the photonic PCR thermal cycler 40 shown in FIG. 3, the light fromthe LEDs 22 is continuous-wave and randomly polarized. Therefore, theefficiency of light-to-heat conversion would be lower in this case thana pulsed laser, because as electrons are excited to higher energystates, the probability of further excitation decreases. Despite thepossibility of lower light-to-heat conversion efficiency than a pulsedlight source, however, LEDs require minimal power consumption and areextremely low in cost compared to laser sources, making LEDs an idealPCR heating source for POC testing. The component cost of theinstruments for our ultrafast photonic PCR thermal cycle can befabricated for less than US $100, including the LEDs, focus lens anddriver, with the Labview (as part of application software 48) and dataacquisition board for temperature control integrated into amicrocontroller module (e.g. computing module 42 in FIG. 3).

The maximum power consumption of an LED is generally around 3.5 W at 1 Ainjection current. FIG. 5B shows the temperature profiles of a 10 μLvolume of solution (here, glycerol was used to show maximum heatingtemperature) with different thickness thin Au films at a fixed injectioncurrent of 500 mA. The maximum temperatures are increased as thethickness of thin Au film increases from 10 nm to 120 nm due to theincreasing optical absorption.

The photothermal heating of the 120 nm-thick Au film was furthercharacterized as a function of injection current as shown in FIG. 5C,because the heating rate is determined by the amount of dissipated power(i.e., an injection current of LEDs). FIG. 5D summarizes the temperatureof a solution after 3 min heating with different thickness Au films andvarying injection current (see Table 2). These results clearly indicatethat the maximum temperatures are increased with an increase of Au filmthickness to 120 nm and an increase of injection current to 1 A.

Complete PCR thermal cycling, consisting of 3 representativetemperatures (94° C. for denaturation, 60° C. for annealing, and 72° C.for extension), is demonstrated using an LED-driven photonic PCR thermalcycler, as shown in FIG. 5E. To prevent evaporation during thermalcycling, 10 μL of PCR buffer was covered with 30 μL of mineral oil. Theaverages and standard deviations at each temperature were obtained fromthe temperature profile and the results are 94.09±0.17° C. at 94° C.,59.94±0.13° C. at 60° C. and 72.02±0.12° C. at 72° C., respectively,showing excellent temperature accuracy and stability. The initialtemperature fluctuation before reaching setting temperature may befurther reduced by optimizing the proportional-integral-derivative (PID)controller value in Labview.

f. Ultrafast Thermal Cycling and Nucleic Acid Amplification

In order to determine maximum heating and cooling rates, a thermal cyclewas performed, whereby the solution (here, 5 μL of PCR mixture coveredwith 30 μL of mineral oil) temperature was rapidly cycled between 55° C.and 95° C. The temperature range mirrors the same denaturation (95° C.)and annealing (55° C.) temperatures.

FIG. 6A through FIG. 6C show plots and images of ultrafast thermalcycling and DNA amplification. FIG. 6A is a plot of representativetemperature profiles of 30 ultrafast photonic PCR thermal cycles from95° C. (denaturation) to 55° C. (annealing and extension). The 5 μL ofPCR buffer was covered with 20 μL of mineral oil to prevent evaporationduring thermal cycling. FIG. 6B shows plot of heating and cooling ratesobtained from the ultrafast photonic thermal cycling. FIG. 6C is animage showing formation of product from the photonic PCR thermal cyclerin comparison with a bench-top thermal cycler using a λ DNA template.Heating and cooling rate were 12.79±0.93° C. sec⁻¹ and 6.6±0.29° C.sec⁻¹, respectively.

Referring to FIG. 6A, the ultrafast photonic 30 cycles within 5 minutesare shown. Using the thermal cycling result, heating and cooling rateswere calculated by measuring the temperature difference betweensuccessive temperature maxima and minima, then dividing by the timeinterval between them. The average rates and sample standard deviationswere obtained as shown in FIG. 6B. The average heating and cooling ratesobtained are 12.79±0.93° C. sec⁻¹ and 6.6±0.29° C. sec⁻¹, respectively.The amplification of λ-DNA was performed to verify our photonic PCRmethod.

After running PCR reactions as shown in FIG. 6C, the amplicons werevisualized by E-Ge 2% agarose gels with SYBR Safe. Lane 1 represents the1 kb DNA marker, lane 2 and 3 are the PCR product from ultrafastphotonic PCR with different cycle numbers (94° C. for 0 sec, 62° C. for0 sec), lane 4 and 5 contain positive controls produced from a standardthermal cycle condition (94° C. for 1 sec, 62° C. for 1 sec, 60 cycles)by a bench-top thermocycler (Bio-Rad C1000 Thermal Cycler). A singlemajor band (98 bp) was detected near 100 bp in photonic PCR (Lane 2 and3). This indicates that the λ-DNA was successfully amplified using theultrafast photonic PCR method and system of the present description. Theweak band intensity from the PCR product amplified by photonic PCR couldbe attributed to the lower amplification efficiency compared to atraditional bench-top thermal cycler. Currently, only thin Au film actsas a 2-dimensional photothermal heater, leading to a temperaturegradient of the solution, leading to potentially lower amplificationefficiency of PCR. This limitation can be improved by utilizing a3-dimensional substrate in the PCR chamber for uniform photothermalheating of PCR mixture. Amplification time as well as reagentconsumption could be further reduced, simultaneously improving theefficiency of the PCR reaction by faster molecular diffusion and uniformsolution temperature.

FIG. 8 shows normalized light emission spectra measured from 3 differentLEDs with peak wavelengths of 450 nm (Blue I), 480 nm (Blue II) and 530nm (Green), respectively. Blue I was generated with Luxeon Rebel royalblue star LEDs with a peak wavelength of 447.5 nm, 890 mW at 700 mAinjection current. Blue II was generated with Luxeon Rebel blue starLEDs with a peak wavelength of 470 nm, 70 lm at 700 mA injectioncurrent. Green was generated with Luxeon Rebel green star LEDs with apeak wavelength of 530 nm, 161 Im at 700 mA injection current. Thespectra were measured using Ocean Optics USB 2000+ spectrophotometer.

FIG. 9A and FIG. 9B show transmittance and reflectance spectra,respectively, of the thin Au films on PMMA substrate with differentthickness. Transmittance and reflectance spectra were measured using aShimadzu UV-3101PC UV-Vis-NIR spectrophotometer. An integrating spherewas employed to measure diffuse reflection as well as specularreflection.

FIG. 10 is a plot showing a comparison of 31 ultrafast thermal cyclesfrom 62° C. to 94° C. with and without cooling fan to show the effect ofcooling fan to further reduce the power consumption of photonic PCRthermal cycler.

Example 2

FIG. 11 shows the schematics of experimental set up 50 for LED drivenplasmonic heating of thin Au films. Blue LEDs 22 with 447.5 nm peakwavelength were used for light illumination and the light from LEDs wasfocused by commercially available plastic lens 32. The thin Au films 20with nanometer sized grain were deposited by an electron beamevaporation method. The surface temperature of liquid 16 was measured bylong wavelength infrared (LWIR) camera 52.

FIG. 12 shows the demonstration of fast thermal cycle using 2 μL ofglycerol from 50° C. to 90° C. for 9 min using LED driven plasmonicheating of thin Au films. The injection current was 1 A, and thethickness of thin Au film was 56 nm.

FIG. 13A shows the temperature changes of liquid with different thin Aufilms thickness. As the thickness of Au film increase, the maximumtemperature of liquid is increased, as shown in FIG. 13B. Thetemperature of liquid was rapidly decreased after turn off of LEDs.

FIG. 13C shows the absorbance spectra of thin Au films with differentthickness. The absorption peak around 440 nm is well matched with peakwavelength of LEDs. As the thickness of Au film increase, the absorbanceis also increased, resulting in the increase of maximum temperature.FIG. 13D and FIG. 13E show images of the Au film 20 at 56 nm and 96 nm,respectively.

FIG. 14A shows the temperature changes of liquid with differentinjection current. The thickness of Au film is 64 nm. As the injectioncurrent increase, the maximum temperature is increased as shown in FIG.14B. Therefore, the temperature can readily be controlled by simplychanging the injection current of LEDs. The maximum ramp rate iscalculated at each injection current and it reaches up to 7° C./sec witha 1 A injection current.

FIG. 15 shows the temperature stability of LED driven plasmonic heatingof thin Au films. The 50° C. and 90° C. are representative annealing anddenaturation temperatures, respectively, for a fast 2-step PCR setup.The temperature changes at 50° C. and 90° C. are ±0.5 and ±0.7° C.,showing good temperature stability.

FIG. 16 and FIG. 17 illustrate an exemplary PCR using LED drivenplasmonic heating of thin Au films. The thickness of Au film is 64 nm.The detailed PCR condition is described in figure. FIG. 16 showstemperature profiles of thermal cycling measured by IR camera during PCRreaction. The temperature profile measured by IR camera is a littledifferent, because the IR camera measures only the surface temperatureof liquid. FIG. 17 shows that the LED driven plasmonic thermal cyclerthrough this invention shows comparable nucleic acids amplificationproduct to conventional bench top thermal cycler.

In summary, a novel ultrafast photonic PCR system is disclosed thatutilizes plasmonic photothermal heating of thin Au films driven by LEDs.A thin Au film-based light-to-heat converter was designed and fabricatedto heat a PCR solution over 150° C. by harnessing gold plasmon-assistedhigh optical absorption. Ultrafast thermal cycling from 55° C.(annealing) to 95° C. (denaturation) was achieved within 5 minutes for30 cycles with ultrafast heating (12.79±0.93° C. sec⁻¹) and cooling(6.6±0.29° C. sec⁻¹) rates. Nucleic acid (λ-DNA) amplification using ourultrafast photonic PCR thermal cycler was successfully demonstrated. Thesystems and methods of the present description are shown to provide asimple, robust and low cost photonic PCR technique, with ultrafastthermal cycling capability, which is ideal for POC moleculardiagnostics, having the following beneficial attributes: 1)affordability (less expensive system with a LED and lens); 2)portability(compact and light PCR system without a heating block); 3)simplicity (use of use with disposable PCR chip); 4) user-friendlyinterface with LED driver and display; 5) rapid and robust PCR withoutenvironmental stress; 6) generally equipment free—only LED andmicrocontroller modules may be required and incorporated with use ofcell-phone camera; and 7) durability in harsh environments & low powerconsumption. While the tested set-up was based on only one PCR well,integration of multiple wells and an array of LED's is contemplated toallow for high-throughput and multiplexed amplification, as well asoptimizing the PCR reaction chamber for uniform heating.

Embodiments of the present technology may be described with reference toflowchart illustrations of methods and systems according to embodimentsof the technology, and/or algorithms, formulae, or other computationaldepictions, which may also be implemented as computer program products.In this regard, each block or step of a flowchart, and combinations ofblocks (and/or steps) in a flowchart, algorithm, formula, orcomputational depiction can be implemented by various means, such ashardware, firmware, and/or software including one or more computerprogram instructions embodied in computer-readable program code logic.As will be appreciated, any such computer program instructions may beloaded onto a computer, including without limitation a general purposecomputer or special purpose computer, or other programmable processingapparatus to produce a machine, such that the computer programinstructions which execute on the computer or other programmableprocessing apparatus create means for implementing the functionsspecified in the block(s) of the flowchart(s).

Accordingly, blocks of the flowcharts, algorithms, formulae, orcomputational depictions support combinations of means for performingthe specified functions, combinations of steps for performing thespecified functions, and computer program instructions, such as embodiedin computer-readable program code logic means, for performing thespecified functions. It will also be understood that each block of theflowchart illustrations, algorithms, formulae, or computationaldepictions and combinations thereof described herein, can be implementedby special purpose hardware-based computer systems which perform thespecified functions or steps, or combinations of special purposehardware and computer-readable program code logic means.

Furthermore, these computer program instructions, such as embodied incomputer-readable program code logic, may also be stored in acomputer-readable memory that can direct a computer or otherprogrammable processing apparatus to function in a particular manner,such that the instructions stored in the computer-readable memoryproduce an article of manufacture including instruction means whichimplement the function specified in the block(s) of the flowchart(s).The computer program instructions may also be loaded onto a computer orother programmable processing apparatus to cause a series of operationalsteps to be performed on the computer or other programmable processingapparatus to produce a computer-implemented process such that theinstructions which execute on the computer or other programmableprocessing apparatus provide steps for implementing the functionsspecified in the block(s) of the flowchart(s), algorithm(s), formula(e),or computational depiction(s).

It will further be appreciated that the terms “programming” or “programexecutable” as used herein refer to one or more instructions that can beexecuted by a processor to perform a function as described herein. Theinstructions can be embodied in software, in firmware, or in acombination of software and firmware. The instructions can be storedlocal to the device in non-transitory media, or can be stored remotelysuch as on a server, or all or a portion of the instructions can bestored locally and remotely. Instructions stored remotely can bedownloaded (pushed) to the device by user initiation, or automaticallybased on one or more factors. It will further be appreciated that asused herein, that the terms processor, computer processor, centralprocessing unit (CPU), and computer are used synonymously to denote adevice capable of executing the instructions and communicating withinput/output interfaces and/or peripheral devices.

From the description herein, it will be appreciated that that thepresent disclosure encompasses multiple embodiments which include, butare not limited to, the following:

1. An apparatus for nucleic acids amplification, comprising: a supportplatform comprising one or more wells configured to hold a sample; aplasmonic thin film disposed within the one or more wells; and a lightsource; wherein the light source is configured to be directed at theplatform such that exposed light from the light source to generatesplasmonic photothermal light-to-heat conversion within the plasmonicthin film and subsequent heating of the sample.

2. The apparatus of any preceding embodiment, further comprising: a lensdisposed between the light source and the support platform; wherein thelens is configured to focus the exposed light at the one or more wells.

3. The apparatus of any preceding embodiment, further comprising atemperature sensor configured to monitor the temperature of the sample.

4. The apparatus of any preceding embodiment, further comprising: acontroller coupled to one or more of the light source and temperaturesensor; the controller configured for controlling one or more of dataacquisition from the temperature sensor and actuation of the lightsource.

5. The apparatus of any preceding embodiment, wherein the controller isconfigured for controlling actuation of the light source to modify oneor more of exposure duration and injection current at the plasmonic thinfilm.

6. The apparatus of any preceding embodiment, wherein the plasmonicthin-film sheet comprises a nanometer sized grain to enhance lightabsorption through surface plasmon resonance.

7. The apparatus of any preceding embodiment, wherein the platformcomprises a translucent or transparent polymer.

8. The apparatus of any preceding embodiment, wherein the temperaturesensor comprises a long wavelength infrared (LWIR) camera orientedadjacent to the sample.

9. The apparatus of any preceding embodiment, further comprising adiffuser associated with the focusing lens to evenly distribute theexposed light to the plasmonic thin film.

10. The apparatus of any preceding embodiment, wherein the light sourcecomprises one or more LED's having a wavelength selected for maximumlight absorption within the plasmonic thin film.

11. The apparatus of any preceding embodiment, further comprising: adigital camera, photodiode or spectrophotometer for the real-timedetection of nucleic acids within the sample.

12. The apparatus of any preceding embodiment, wherein the platformcomprises 2D or 3D microstructures or nanostructures in the form of oneor more of a pillar array, 1D or 2D grating, photonic crystal,hemisphere.

13. A method for nucleic acids amplification, comprising: disposing afluid sample within the one or more wells having a plasmonic thin film;directing a light source at the plasmonic thin film to generateplasmonic photothermal light-to-heat conversion within the plasmonicthin film; and heating the sample as a result of the light-to-heatconversion within the plasmonic thin film.

14. The method of any preceding embodiment, further comprising: focusinglight from the light source at the one or more wells.

15. The method of any preceding embodiment, further comprising:monitoring the temperature of the sample.

16. The method of any preceding embodiment, further comprising:controlling one or more of data acquisition from a temperature sensorand actuation of the light source.

17. The method of any preceding embodiment, wherein controllingactuation of the light source comprises controlling one or more ofexposure duration and injection current at the plasmonic thin film.

18. The method of any preceding embodiment, wherein the plasmonicthin-film sheet comprises an Au film with a nanometer sized grain toenhance light absorption through surface plasmon resonance.

19. The method of any preceding embodiment: wherein the one or morewells are formed in a translucent or transparent platform; wherein thelight from the light source is directed through at least a portion ofthe platform to the plasmonic thin film.

20. The method of any preceding embodiment, further comprising:diffusing the focused light to evenly distribute the light to theplasmonic thin film.

21. The method of any preceding embodiment, wherein the light is emittedat a wavelength selected for maximum light absorption within theplasmonic thin film.

22. The method of any preceding embodiment, further comprising:detecting a fluorescence signal within the sample.

23. A plasmonic heater apparatus for nucleic acids amplification,comprising: a substrate having a plurality of reaction wells configuredfor holding a sample; wherein a surface of each of the plurality ofreaction wells is covered with a plasmonic thin film; and a light sourcedirected at the substrate; the light source configured to illuminate theplasmonic thin film at a wavelength and duration that causesphotothermal heating of the plasmonic thin film and subsequent heatingof the sample.

24. The apparatus of any preceding embodiment, further comprising: atleast one temperature sensor configured to monitor the temperature ofthe sample in each well.

25. The apparatus of any preceding embodiment, further comprising: (a) acontrol module coupled to the temperature sensor and the light source;(b) the control module comprising a processor and a memory storinginstructions executable on the processor; (c) said instructions, whenexecuted by the processor, performing steps comprising: (i) monitoringsample temperature; and (ii) actuating the light source at a frequencyand duration to produce selected sample temperatures over time.

26. The apparatus of any preceding embodiment, wherein said instructionswhen executed by the processor further perform steps comprisingdetecting or a fluorescence signal within the sample.

27. The apparatus of any preceding embodiment, wherein the substratecomprises a transparent or translucent polymeric such that the reactionwells are formed in the sheet as a digital microfluidic array.

28. The apparatus of any preceding embodiment: wherein with a surface ofeach well is covered with nanoplasmonic structures; and wherein thelight source configured to illuminate the nanoplasmonic structures onthe surface of the wells at a resonance wavelength of nanoplasmonicstructures and duration that causes plasmonic photothermal heating ofthe nanoplasmonic structures.

29. The apparatus of any preceding embodiment, wherein the controlmodule is configured to control the light source for ultrafast thermalcycling for portable multiplexed PCR at low power consumption.

Although the description herein contains many details, these should notbe construed as limiting the scope of the disclosure but as merelyproviding illustrations of some of the presently preferred embodiments.Therefore, it will be appreciated that the scope of the disclosure fullyencompasses other embodiments which may become obvious to those skilledin the art.

In the claims, reference to an element in the singular is not intendedto mean “one and only one” unless explicitly so stated, but rather “oneor more.” All structural, chemical, and functional equivalents to theelements of the disclosed embodiments that are known to those ofordinary skill in the art are expressly incorporated herein by referenceand are intended to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed as a “means plus function”element unless the element is expressly recited using the phrase “meansfor”. No claim element herein is to be construed as a “step plusfunction” element unless the element is expressly recited using thephrase “step for”.

TABLE 1 Materials Parameters For Electromagnetic Simulation Density HeatCapacity Thermal Conductivity ρ(kg m⁻³) C(J kg⁻¹ K⁻¹) k(W m⁻¹ K⁻¹) Gold19,300 129 317 PMMA 1,180 1,420 1.93 Water 998 4,180 0.6

TABLE 2 Temperature As A Function Of Film Thickness And InjectionCurrent Thickness (nm) 10 nm 20 nm 40 nm 80 nm 120 nm Current (mA) MeanStd. Mean Std. Mean Std. Mean Std. Mean Std. 100 35.8 0.71 37.8 1.1340.7 0.72 45.1 0.66 45.7 .53 200 47.3 1.41 51.6 1.33 55.5 1.70 65.7 1.1466.0 0.73 300 58.8 2.47 64.3 1.79 74.4 1.03 84.0 1.34 84.5 1.22 400 69.03.89 75.9 2.54 89.3 1.74 101.9 2.26 102.6 2.71 500 78.4 4.07 86.8 3.03103.1 3.91 116.8 1.06 119.5 1.61 600 87.1 5.13 97.4 3.98 115.1 3.15126.9 1.39 128.3 2.47 700 95.4 5.13 108.9 4.34 123.9 3.57 133.7 3.07134.9 3.70 800 104.5 4.24 117.4 5.27 131.8 5.14 139.7 4.50 139.7 4.58900 111.9 3.36 124.1 6.00 137.2 5.38 145.3 4.17 145.8 5.83 1000 118.94.77 128.1 5.84 142.0 5.29 150.1 4.20 151.5 5.29

What is claimed is:
 1. A method for nucleic acids amplification,comprising: disposing a sample within one or more wells comprising athin film; directing a light source at the thin film to generatelight-to-heat conversion within the thin film; and heating the sample asa result of light-to-heat conversion within the thin film.
 2. The methodof claim 1, further comprising: focusing light from the light source atthe one or more wells.
 3. The method of claim 1, further comprising:monitoring a temperature of the sample.
 4. The method of claim 1,further comprising: controlling one or more of data acquisition from atemperature sensor and actuation of the light source.
 5. The method ofclaim 1, wherein controlling actuation of the light source comprisescontrolling one or more of exposure duration and injection current atthe thin film.
 6. The method of claim 1, wherein the thin film comprisesan Au film with a nanometer sized grain to enhance light absorptionthrough surface plasmon resonance.
 7. The method of claim 1, wherein theone or more wells are formed in a translucent or transparent platform;and wherein the light from the light source is directed through at leasta portion of the platform to the thin film.
 8. The method of claim 1,further comprising: diffusing focused light to evenly distribute thelight to the thin film.
 9. The method of claim 1, wherein the light isemitted at a wavelength selected for maximum light absorption within thethin film.
 10. The method of claim 1, further comprising: detecting afluorescence signal within the sample.